2.1. Bacterial Enzymology
The emergence of resistance to existing antibiotics has rejuvenated interest in bacterial enzymology. It is hoped that detailed mechanistic and structural information about bacterial enzymes involved in critical biosynthetic pathways could lead to the development of new antibacterial agents. Some of the best antibiotics function by interfering with the biosynthesis of the peptidoglycan polymer that surrounds bacterial-cells. Because interference with peptidoglycan biosynthesis is a proven strategy for treating bacterial infections, all of the enzymes involved in peptidoglycan biosynthesis are potential targets for the development of new antibiotics. Although remarkable progress has been made in characterizing some of the early enzymes in the biosynthetic pathway (See, e.g., Fan, C. et al. Science 1994, 266, 439; Benson, T. E. et al. Nat. Struct. Biol. 1995, 2, 644; Jin, H. Y. et al. Biochemistry 1996, 35, 1423; Skarzynski, T. et al. Structure 1996, 4, 1465; Schonbrunn, E. et al. Structure 1996, 4, 1065; Benson, T. E. et al. Biochemistry 1997, 36, 806), the downstream enzymes have proven exceedingly difficult to study.
There are two main reasons for this difficulty: First, the downstream enzymes are membrane-associated, making them intrinsically hard to handle (See, e.g., Gittins, J. R. et al. FEMS Microbiol. Rev. 1994, 13, 1; Bupp, K. and van Heijenoort, J. J. Bacteriol. 1993, 175, 1841.); second, discrete substrates for most of the downstream enzymes are either not available or not readily so (See, e.g., Pless, D. D. and Neuhaus, F. C. J. Biol. Chem. 1973, 248, 1568; van Heijenoort, Y. et al. J. Bacteriol. 1992, 174, 3549.).
For example, the Lipid II substrate is difficult to obtain in large quantities from natural sources and difficult to handle due to its detergent like properties. In the absence of readily available, well-behaved discrete substrates, it has been impossible to develop enzyme assays that can be used to measure the activity of the downstream enzymes reliably and under a well-defined set of reaction conditions. This unfulfilled need has thwarted attempts to purify many of the downstream enzymes in an active form suitable for structural characterization and attempts to obtain detailed mechanistic information on such enzymes. It has also complicated screening for inhibitors. The use of synthetic or semi-synthetic substrate analogs with characteristics that make them easier to handle than the natural substrates and/or facilitate product detection could permit the study of many downstream enzymes (Men, H. et al. J. Amer Chem Soc 1998, 120, 2485 and PCT publication WO 99/38958).
2.2 Peptidoglycan Biosynthesis
FIG. 1 illustrates the key pathways for biosynthesis of peptidoglycan. Lipid I is converted to Lipid II by the enzyme MurG (N-acetylglucosaminyltransferase). Several reactions occur downstream from the MurG-catalyzed reaction. After translocation, Lipid II is either conjugated to another Lipid II or to an existing peptidoglycan by the enzyme transglycosylase. Cross-linking of peptides of spatially adjacent peptidoglycan molecules is accomplished by the enzyme transpeptidase.
2.3. Transglycosylases
FIG. 2 illustrates one family of downstream enzymes, the transglycosylases, which are involved in the late steps of peptidoglycan biosynthesis. Transglycosylases catalyze an extracellular step in the biosynthetic pathway of peptidoglycan biosynthesis, i.e., the coupling of two Lipid II molecules, coupling of two Lipid II analogs, or the coupling of one Lipid II molecule to the C4 hydroxyl of an N-acetylglucosaminyl acceptor that is part of the growing peptidoglycan polymer.
FIG. 3 illustrates the cellular location of the key enzymes of peptidoglycan biosynthesis. In the cytoplasm MurG acts to form Lipid I and transglycosylase acts to form Lipid H. The transglycosylase and transpeptidase reactions occur extracellularly, at the bacterial membrane surface.
There are multiple different transglycosylases in bacterial cells. Both bifunctional and monofunctional enzymes have been identified (Nakagawa, J. et al. J. Biol. Chem., 1984, 259, 13937; Spratt, B. G. et al. Mol. Microbiol., 1996, 19, 639). The bifunctional enzymes also contain transpeptidase activity which is sensitive to penicillin. The bifunctional enzymes are most commonly known as penicillin binding proteins (PBPs). The transglycosylase domains of some PBPs are believed to be functional in the absence of other proteins (Vollmer, W. J. Biol. Chem. 1999, 279, 6726). The transglycosylase domains of other PBPs are believed to be dependent on the presence of other proteins that have been implicated in bacterial cell growth or cell division (Vollmer, ibid). It is known that inhibition of transglycosylase activities, e.g., by treatment with moenomycin, leads to bacterial cell death. Moenomycin besides being known as antibiotic is also as an antitumor drug, see for example incorporated by reference U.S. Pat. No. 4,011,140. Antibiotics are also important in food industry as growth-promoting agents for plants and domestic animals, e.g., U.S. Pat. No. 3,949,070 discloses pholipomycin for such an application. Hence, the instant transglycosylase assay is an important tool for identifying antibiotics and other classes of drugs like antitumor drugs, antiviral drugs, growth promoters, etc. Unfortunately, there is only one membrane-free assay for transglycosylase activity. This assay involves the isolation of [14C]-radiolabeled Lipid II from bacterial cells or bacterial membrane preparations supplemented with appropriate starting materials (van Heijenoort, Y. et al. FEBS Lett., 1978, 89, 141). The isolated Lipid II is then treated with a crude or a partially purified preparation of a PBP and the formation of polymer is detected by the appearance of radioactivity at the baseline of a paper chromatogram or by retention of radioactivity on a filter. This assay has severe limitations. The major one is that it has only been shown to work for a membrane bound form of PBP1b of E. coli origin (Di Giulmi, A. M. et al. J. Bacteriol 1998, 180, 5652). In addition, the Lipid II substrate is difficult to isolate in significant quantities and the assay can be hard to reproduce due to problems handling the substrate, which contains a 55-carbon lipid chain.
FIG. 4 illustrates some of the difficulties in isolating and handling Lipid II, and the problems in detecting the formation of products smaller than polymers. These problems have prevented the development of simple, direct assays for transglycosylase activity that work for both soluble and membrane-bound transglycosylase domains. Consequently, it has not been possible to demonstrate activity in transglycosylase domains that have been engineered for solubility or to purify any transglycosylase domain to homogeneity in a quantifiably active form or to determine the minimal functional length of the transglycosylase domain; nor has it been possible to carry out any detailed mechanistic studies, or to determine the substrate requirements.
Development of reliable, direct assays for monitoring transglycosylase activity that facilitates the detection of products of any length would promote the development of new antibiotics by: a) permitting the purification of active transglycosylase domains suitable for structural and mechanistic investigations; and b) providing a screen for compounds that inhibit transglycosylase activity. There are no direct assays for monitoring transglycosylase activity that utilize synthetic substrate analogs with solubility properties that differ from the natural substrates (which contain very long lipid chains and are not soluble in water). Furthermore, there are no direct assays for monitoring transglycosylase activities that are capable of detecting non-polymeric products. Finally, there are no direct assays for monitoring transglycosylase activities that make use of purified Lipid II or Lipid I substrate analogs which further contain a fluorophore, chromophore, luminophore, or affinity label (e.g., lectin) to facilitate detection of product.
Previous assays for bacterial transglycosylase activity required radiolabeling and purification of the endogenous Lipid II substrate, N-acetylglucosamine-β-1,4-MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (Brotz, H. et al. Mol Microbiol., 1998, 30, 317; Esteve-Garcia, E. et al. Poult Sci., 1997, 76, 1728; Brotz, H. et al. Eur J Biochem., 1997, 246, 193; Pan, Y. T. and Elbein, A. D. Arch Biochem Biophys., 1996, 335. 258: Mohan, B. et al. Br Poult Sci., 1996, 37, 395; Izat, A. L. et al. Poult Sci., 1990, 69, 1787: Salmon, R. E. and Stevens, V. I. Poult Sci., 1990, 69, 1133; Jiraphocakul, S. et al. Poult Sci. 1990, 69, 1966; Zotchev, S. B. et al. Dokl Akad Nauk SSSR., 1990, 313, 203; Mani, N. et al. J Antibiot (Tokyo), 1998, 51, 471; Mani, N. et al. J Antibiot (Tokyo) 1998, 607, 11; van Heijenoort, Y. et al. J Bacteriol., 1992, 174, 3549; van Heijenoort, Y. et al. J Bacteriol, 1992, 174, 6004). These methodologies involve multiple purification steps, yield limited amounts of Lipid II, and require radiolabel for detection of transglycosylase product. In addition, because of the insoluble nature of the Lipid II substrate in an aqueous milieu, irreproducible activity is also a problem. Separation of product from substrate in those methods is performed by paper chromatography or trapping product on filters.
Therefore, there exists a need for direct and simple enzyme assays that can be used both for effective screening of enzyme inhibitors and for the purification, characterization and identification of transglycosylase, its various mutants and active fragments thereof.